1. Field of the Invention
The present invention relates, in general, to a fluorescence microscope and method of observing samples using the microscope and, more particularly, to a fluorescence microscope and method of observing samples using the microscope, which can reduce optical noise and obtain images with higher sensitivity, thus obtaining precise information about the density, quantity, location, etc. of a fluorophore, and which can simultaneously process separate images even when a plurality of fluorophores having different excitation and fluorescent wavelength ranges is distributed, thus easily obtaining information about the fluorophores.
The present invention relates, in general, to a fluorescence microscope, which is an optical instrument, and more particularly, to technology used to observe micro-samples in detail using fluorescence in a variety of biomedical fields, including the fluorescence analysis of a biochip, such as a DNA microarray. A fluorescence microscope is a device for irradiating light onto a micro-object, executing an excitation and fluorescence emission process on the micro-object using the irradiated light, capturing emitted fluorescence, and observing information, such as the image of the micro-object.
2. Description of the Related Art
As shown in FIG. 1, a conventional fluorescence microscope separates the paths of excitation light 12 and fluorescent emission 13 using a beam splitter that is implemented with a combination of an excitation filter 10 installed in front of a light source 5, shielding filters and a dichroic mirror 15. Since an interference coating film on the surface of the dichroic mirror 15 has characteristics of efficiently reflecting the excitation light 12 and allowing the fluorescence emission 13 having a longer wavelength to efficiently pass therethrough, the excitation light 12 cannot be efficiently transferred in the direction of a Charge Coupled Device (CCD) camera 40, and the fluorescent emission 13, emitted from a micro-object existing in a sample 25, is mainly transferred in the direction of the CCD camera 40. Since such a light irradiation scheme uses fluorescence having a unique wavelength emitted from the micro-object, the light irradiation scheme can obtain an image having improved contrast compared to a conventional scheme of principally irradiating light onto an object through a condenser of a microscope and observing light scattered from the object, and has been widely used in typical fluorescence microscopes.
However, as shown in FIG. 1, a portion where the paths of the excitation light 12 and the fluorescence emission 13 are spatially identical to each other exists in front of and behind an objective lens 20, so that optical noise 55, 50 and 45 is caused due to the autofluorescence generated by optical components of the objective lens 20, and the formation of hot spots caused by scattering sources or fluorescence generation sources existing inside and on the objective lens 20. These optical noises decrease the contrast of a formed fluorescence image, and causes difficulty in the precise registration of weak fluorescence, generated due to a small amount of fluorophore present in the micro-object to be observed.
Further, as in the case of the conventional fluorescence microscope, in the construction employing a component (for example, the above-described dichroic mirror) 15 for selecting an optical spectrum on the light path so that the excitation light 12 and the fluorescence emission 13 pass through the same path in front of and behind the objective lens 20 and, in that state, the excitation light 12 is prevented from reaching the observation unit 40, such as a CCD camera, it is basically impossible to simultaneously perform observation, such as the comparison of samples colored with polychromatic dyes, on a single screen.
In order to solve this problem, as shown in FIG. 2, a scheme of preventing the excitation light 12 from passing through the objective lens 20 and separating the spatial paths of the excitation light 12 and the fluorescence emission 13 has been proposed (for example, the biochip reader “Array WoRx” by Applied Precision of U.S. that employs a scheme of combining a light source 5 and an optical fiber 60 to irradiate light onto an analysis object). As shown in FIG. 2, such a scheme irradiates light onto an object at an angle oblique to an optical axis direction of the objective lens 20, thus preventing the excitation light 12 from passing through the objective lens 20 and reducing the amount of optical noise. However, in this scheme, a considerable available space S is required between the objective lens 20 and the object 25, which causes the use of an objective lens 20 having a high numerical aperture to be difficult. Moreover, there is a problem in that light scattered from the object 25 (including dust) and several surfaces of the microscope is incident on the objective lens 20, so that it is difficult to prevent the occurrence of optical noise.
FIG. 3 illustrates Total Internal Reflection Fluorescence Microscopy (TIRFM), which irradiates excitation light 140 using total internal reflection. In this method, the paths of the excitation light 140 and fluorescence emission are separated using total reflection occurring when an incident angle exceeds a critical angle at the interface between two media. Further, an evanescent wave generated from a medium having low optical density is used for the fluorescence excitation of micro-objects 110 arranged around an interface.
In this construction, as shown in FIG. 3, observation objects are placed on a medium having a refractive index of n1. The fluorescence microscope is not different from the above-described fluorescence microscope in that the observation objects 110 include fluorophores and the fluorophores emit fluorescence due to the behavior of excitation light. The emitted light passes through a second medium and is directed to the interface between the first and second media at an incident angle of θ. It is well known that, if the reflective indices of the second and first media satisfy a relationship of n2>n1, and the incident angle of the light satisfies a relationship of θ>θc=sin−1(n1/n2) (where θc is a critical incident angle), Total Internal Reflection (TIR) occurs at the interface between the first and second media. The light reflected from the interface completely returns to the second medium.
A slight part of electromagnetic radiation incident on the interface between the two media passes through the interface, so that the intensity thereof exponentially attenuates in the z axis direction of FIG. 3 and the slight part is extinguished and cannot be propagated into the first medium. Therefore, the propagation of the evanescent wave, the intensity of which decreases exponentially, occurs in an extremely small region 130 around the interface of the first medium. If the interface of the first medium is expressed by z=0, the intensity of the evanescent wave obtained in the z axis direction is given by the following equation.I(z)=I0e−z/d, where d=(λ0/4π)(n22sin θ−n12) −1/2
The evanescent wave can function to excite the fluorophores present in the micro-objects 120 existing around the interface, and the penetration depth of the evanescent wave into the first medium generally does not exceed several hundred nanometers. Therefore, this method is suitable for the observation of micro-objects existing in a thin region 130 around the interface. However, since the intensity of the evanescent wave is low as described above, there is a problem in that the sensitivity of detected light is excessively low, and only micro-objects existing around the interface are consistently observed. Accordingly, this method is problematic in that it is not suitable for the observation of micro-objects distributed within a region having a relatively high volume.
Such Total Internal Reflection Fluorescence Microscopy (TIRFM) is implemented with the following two structures: a structure (1) in which light is provided from the arrangement direction of an objective lens to the direction of the interface, and a structure (2) in which light is provided from a direction opposite to the objective lens to the direction of the interface.
The above structure (1) is disclosed in U.S. Patent Application Publication No. 2002-97489, and depicted in FIG. 4. Such a structure is designated as an inverted microscope, in which the rearmost lens 151 of objective lenses, a sample 145, a cover glass 147 and immersion oil 149 are shown in FIG. 4. The rearmost lens 151 of the objective lenses is adjacent to the immersion oil 149 having a refractive index of n1.
The sample 145 having a refractive index of n2 is placed on the cover glass 147. Excitation light 140 is incident from a light source (illuminator), passes through the immersion oil 149 and is totally reflected from the interface between the cover glass 147 and the sample 145 through the cover glass 147. The refractive index of the cover glass 147 is almost equal to that of the immersion oil 149. An evanescent wave is generated from the surface of the cover glass 147, and fluorescence is emitted from the molecules of the sample adjacent to the cover glass 147. The emitted fluorescence is focused onto an image processing unit after passing through a dichromatic filter (not shown) and an emission filter (not shown) that are arranged below the objective lens 151.
However, the above-described fluorescence observation system includes the immersion oil 149, and requires the very complicated objective lens 151 having a high numerical aperture. Further, since the above fluorescence observation system requires components such as a color filter for selecting an optical spectrum, it is impossible to perform an operation of simultaneously processing separate florescent images using color images and obtaining information about the separate images in the case where a plurality of fluorophores having different excitation and fluorescence wavelength ranges are distributed. Further, since the irradiation of excitation light and the collection of fluorescence emission are performed by the same objective lens 151, optical noise is caused due to the autofluorescence generated by the components of the objective lens 151, and the hot spots caused by scattering sources, etc. distributed inside and on the objective lens 151, thus deteriorating the sensitivity of a microscope.
The above structure (2) is disclosed in U.S. Pat. No. 6,255,083, and depicted in FIG. 5. The microscope uses a gas laser having a tunable wavelength as a light source 201, which allows laser light to pass through a laser line filter 210 so as to filter excitation light only. Thereafter, the laser light is incident on a fused-silica right angle prism 202 placed just below a cover slip 203. Molecules, which are samples to be observed, are immersed in a buffer solution 204 placed on the cover slip 203.
Referring to FIGS. 5 and 6, laser light 240 incident on the prism 202 is refracted and incident at an angle greater than a critical angle between the fused-silica right angle prism 202 and the buffer solution 204. Therefore, the laser light is totally internally reflected (TIR) by the prism 202, and an evanescent wave is generated in a region adjacent to the interface of the buffer solution 204. Fluorescence emissions 255 from the samples 110 are collected by an objective lens 205 immersed in the buffer solution 204, and an image of an object is projected onto a camera 208 by a multi-wavelength viewer 207 for partially separating an image according to wavelength.
The above-described two types of fluorescence microscopes using total internal reflection employ a scheme of exciting observation objects existing within a range of about a monolayer using an evanescent wave, and utilizing fluorescence emission emitted from the observation objects, and are suitable for the observation of micro-areas, thus providing an excellent means for the research of cellular and molecular biology. However, there are problems in that, since the intensity of the evanescent wave is low as described above, the sensitivity of detected light is excessively low, and only micro-objects existing around an interface are consistently observed. Accordingly, the above microscopes have a problem in that they are not suitable for the observation of micro-objects distributed within a region having a relatively high volume.
In addition, the conventional apparatuses for observing micro-objects, such as biochips, have a further problem in that various optical noises must be eliminated and optical filters corresponding to several types of fluorescent dyes having different fluorescence emission wavelengths must be used, thus complicating the observation apparatuses, and unnecessarily increasing the size thereof.